SDS-PAGE of Proteins

SDS-PAGE

Sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (SDS-PAGE)

The method is based on - the separation of proteins according to size

Used – Separation of proteins 

          determine the relative molecular mass of proteins

SDS (CH₃-(CH₂)₁₀-CH₂OSO₃^- Na⁺) → is an anionic detergent

Buffer-

1.            β-mercaptoethanol – reduces/breaks disulphide bonds in protein tertiary structure & converted to linear primary structure
2. 
   
              SDS - denatures the protein
3.        bromophenol blue – is an ionisable tracking dye which use to monitor the electrophoretic run
4.       sucrose or glycerol - gives the sample solution density which helps to settle the sample at the bottom of loading well
5.       Tris- HCl – gives the solution its pH buffering capacity

loading dye - 

1. help to get know when to switch off
2. show how goodly separated.

negatively charged SDS molecules binds for every two amino acid residues in denatured liner polypeptide chain.

2 parts in the gel.

Stacking Gel	Separating/Resolving Gel
1st part of the gel	2nd part of the gel
pH is 6.8	pH is 8.8
Have larger pore size(4% acrylamide)	Have smaller pore size(10-15% acrylamide/polyacrylamide)
Comparatively short ( 0.8cm)	Comparatively long ( 5cm)

stacking gel

stacking gel - concentrate the protein sample into a sharp band before it enters the main separating gel.

This is achieved by using differences in ionic strength and pH between the electrophoresis buffer and the stacking gel buffer.

Negatively increases as follows in stacking gel-

glycinate ions < protein–SDS complexes < chloride ions (Cl^-)

so, in this protein-SDS complex is stacked between glycinate ions & chloride ions (Cl^-). Due to this called as stacking gel

(glycine isoelectric point is 5.95. so, glycinate ions have slight negativity.)

• Conductivity inversely proportional Field strength
• Conductivity  proportional to concentration

Separating/Resolving Gel 

When glycinate reaches the separating gel it becomes more fully ionized in the higher pH environment and its mobility increases

Due to this, negatively increases as follows in separating gel-

protein–SDS complexes< glycinate ions< chloride ions (Cl^-)

then,

•      the smaller the protein -  the more easily it can pass through the pores of the gel, 
•      large proteins - are successively retarded by frictional resistance due to the sieving effect of the gels.
•      Due to this protein are separate on the basis of their size.

•      When the dye reaches the bottom of the gel, the current is turned off
•      Stain with an appropriate stain solution (usually Coomassie Brilliant Blue) and then washed in destain solution
•     The destain solution -  removes unbound background dye from the gel, leaving stained proteins visible as blue bands on a clear background

Gels of 15% polyacrylamide are therefore useful for separating proteins in the range

M_r 100 000 to 10 000

·       A pure protein should give a single band on an SDS–polyacrylamide gel

Glycolysis/EMP Pathway

Glycolysis/EMP

Glycolysis degrade a molecule of glucose in a series of enzyme-catalyzed reactions to give/yield 2 molecules of three-carbon(C3) compound pyruvate of lower free energy. Free energy released from glucose is conserved (use to synthesis) in the form of ATP and NADH.

·       Glycolysis reactions are take place in cytosol.

Major contributors:

1.     Gustav Embden

2.     Otto Meyerhof

3.     Jacob Parnas

Chemical Strategy of Glycolysis is:

1. Add phosphoryl groups to the glucose
2. Chemically convert phosphorylated intermediates into compounds with high phosphate group-transfer potentials.
3. Chemically couple the subsequent hydrolysis of reactive substances to ATP synthesis.

Stages of Glycolysis

Glycolysis has 10 steps which can dived into 2 phases.

• ·       Stage I (Reactions 1–5) - A preparatory stage - hexose glucose is phosphorylated and cleaved to yield 2 molecules of the triose glyceraldehyde-3-phosphate. This process utilizes 2 ATPs in a kind of energy investment 

• ·       Stage II (Reactions 6–10): Payoff phase -

The 2 molecules of glyceraldehyde-3-phosphate are oxidative converted to pyruvate, with generation of 4 ATPs & 2NADH.so, net ATP gain per glucose molecule in glycolysis is 2.

extra -

Energy Remaining in Pyruvate

Glycolysis releases only a small fraction of the total available energy of the glucose molecule; the two molecules of pyruvate formed by glycolysis still contain most of the chemical potential energy of glucose.

    

Importance of Phosphorylated Intermediates

Each of the 9 glycolytic intermediates between glucose and pyruvate is phosphorylated. The phosphoryl groups appear to have 3 functions -

1. Because the plasma membrane generally lacks transporters for phosphorylated sugars, the phosphorylated glycolytic intermediates cannot leave the cell. After the initial phosphorylation, no further energy is necessary to retain phosphorylated intermediates in the cell, although the large difference in their intracellular and extracellular concentrations 
2. Phosphoryl groups are essential components in the enzymatic conservation of metabolic energy. (extra - Energy released in the breakage of phosphoanhydride bonds (such as those in ATP) is partially conserved in the formation of phosphate esters such as glucose 6-phosphate. High-energy phosphate compounds formed in glycolysis (1,3-bisphosphoglycerate and phosphoenolpyruvate) donate phosphoryl groups to ADP to form ATP)
3. Binding energy resulting from the binding of phosphate groups to the active sites of enzymes lowers the activation energy and increases the specificity of the enzymatic reactions. (extra - The phosphate groups of ADP, ATP, and the glycolytic intermediates form complexes with Mg²⁺ and the substrate binding sites of many glycolytic enzymes are specific for these Mg²⁺ complexes. Most glycolytic enzymes require Mg²⁺ for activity)

Reaction 1 -Phosphorylation of Glucose

• 1^st Reaction of glycolysis is the transfer of a phosphoryl group from ATP to glucose to form glucose-6-phosphate (G6P) in a reaction catalyzed by hexokinase (HK)
• kinases are the enzymes that transfers phosphoryl groups between ATP and a metabolite
• Hexokinase, like many other kinases, requires Mg²⁺ for its activity, because the true substrate of the enzyme is not ATP^4- but the MgATP^2- complex. Mg²⁺ shields the negative charges of the phosphoryl groups in ATP, making the terminal phosphorus atom an easier target for nucleophilic attack by an -OH of glucose

Reaction 2 - Conversion of Glucose 6-Phosphate to Fructose 6-Phosphate

• conversion of G6P to fructose-6-phosphate (F6P) by phosphoglucose isomerase (PGI; also called glucose-6-phosphate isomerase/ phosphohexose isomerase)
• This is the isomerization of an aldose to a ketose

Reaction 3 - Phosphorylation of Fructose 6-Phosphate to Fructose 1,6- Bisphosphate

• phosphofructokinase (PFK) catalyzes the transfer of a phosphoryl group from ATP to fructose 6-phosphate to yield fructose 1,6-bisphosphate(FBP) [previously known as fructose-1,6-diphosphate (FDP)]
• PFK plays a central role in the control of glycolysis because it catalyzes one of the pathway’s rate-determining reactions

Reaction 4 - Cleavage of Fructose 1,6-Bisphosphate

• Aldolase catalyzes the cleavage of FBP to form the 2 trioses glyceraldehyde-3-phosphate (GAP)and dihydroxyacetone phosphate (DHAP)
• Aldol cleavage between C3 and C4 of FBP requires a carbonyl at C2 and a hydroxyl at C4.

Reaction 5- Interconversion of the Triose Phosphates

• DHAP and GAP are ketose–aldose isomers
• Triose phosphate isomerase (TIM or TPI) catalyzes this process

Reaction 6- Oxidation of Glyceraldehyde 3-Phosphate to 1,3-Bisphosphoglycerate

• This involves the oxidation and phosphorylation of GAP to 1,3-bisphosphoglycerate by NAD⁺ and P_i as catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
• aldehyde oxidation, an exergonic reaction, drives the synthesis of the acyl phosphate group at C-1 1,3-bisphosphoglycerate.

Reaction 7- Phosphoryl Transfer from 1,3-Bisphosphoglycerate to ADP

• phosphoglycerate kinase(PGK) transfers the high-energy phosphoryl group from the carboxyl group of 1,3-bisphosphoglycerate to ADP, forming ATP and 3- phosphoglycerate.

Reaction 8- Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate

• phosphoglycerate mutase(PGM) catalyzes the transfer of phosphoryl group between C-2 and C-3 of glycerate which results in conversion of 3PG to 2-phosphoglycerate (2PG).(mutases catalyze the transfer of a functional group from one position to another on a molecule)

Reaction 9 - Dehydration of 2-Phosphoglycerate to Phosphoenolpyruvate

• 2PG is dehydrated to phosphoenolpyruvate (PEP)in a reaction catalyzed by enolase
• The enzyme forms a complex with a divalent cation such as Mg²⁺ before the substrate is bound

Reaction 10 - Transfer of the Phosphoryl Group from PEP to ADP

• Transfer of the phosphoryl group from phosphoenolpyruvate to ADP, catalyzed by pyruvate kinase, which requires K⁺ and either Mg²⁺ or Mn²⁺
• This is a substrate-level phosphorylation

Overall reaction of glycolysis -

Glucose +2NAD⁺ +2ADP +2P_i →

2 pyruvate +2NADH +4H⁺ +2ATP +2H₂O

Regulation of Glycolysis

Enzyme	Inhibitors	Activators
Hexokinase	G-6-P
Phosphofructokinase	ATP, PEP, Citrate	ADP, AMP, Fructose 2,6-P
Pyruvate kinase	ATP

The Oxidizing Power of NAD⁺ Must Be Recycled

• NAD⁺ is the primary oxidizing agent of glycolysis
• The NADH produced by this process must be continually re-oxidized to keep the pathway supplied with NAD⁺

There are three common ways that this occurs -

1. Under anaerobic conditions in muscle, NAD⁺ is regenerated when NADH reduces pyruvate to lactate.
2. Under anaerobic conditions in yeast, pyruvate is decarboxylated to yield CO₂ and acetaldehyde and the latter is reduced by NADH to yield NAD⁺ and ethanol.
3. Under ATPs aerobic conditions, the mitochondrial oxidation of each NADH to NAD⁺ yields 2.5

Thus, in aerobic glycolysis, NADH may be thought of as a “high-energy” compound, whereas in anaerobic glycolysis its free energy of oxidation is dissipated as heat.

References - 

1. Lehninger Biochemistry
2. BIOCHEMISTRY by VOET . D & VOET J.G
3. Bacterial Metabolism by Gerhard Gottschal 
4. MICROBIAL PHYSIOLOGY by Albert G. Moat , John W. Foster & Michael P. Spector

Special thanks - 

Dr. Gagandeep Kaur

DNA microarray

DNA microarray

A DNA microarray also known as DNA chip or a Biochip.

•            Definition - DNA microarrays are solid supports, usually of glass, nylon or silicon, on which Collection of microscopic DNA spots (DNA probes) are immobilized as microdots in an organized grid fashion. Each spot of DNA, called a probe, represents a single gene.

It allows the measurement of the level of gene expression for every gene in genome.

Principle

The principle of DNA microarrays lies on the hybridization between the nucleotide. Using this technology, the presence of one genomic or cDNA sequence in 1,00,000 or more sequences can be screened in a single hybridization.

Hybridization: The property of complementary nucleic acid sequences is to specifically pair

with each other by forming hydrogen bonds between complementary nucleotide base pairs.

Requirements - 

1. DNA chip
2. Target sample
3. Fluorescent dyes
4. Probes
5. Scanner
6. Enzymes

Steps -

1. Sample preparation
2. Purification (Isolate mRNA)
3. Reverse Transcription
4. Labeling
5. Hybridization
6. Scanning
7. Normalization and analysis.

Sample preparation - We’ll use two samples – cancerous human skin tissue & healthy human skin tissue.

Purification (Isolate mRNA) –

•         Extract the RNA from the samples. Using either a column, or a solvent such as phenol-chloroform.
•         isolate the mRNA from total RNA by using affinity column containing beads with Poly-T tails to bind the mRNA. Rinse with buffer to release the mRNA from the beads.

Reverse Transcription & Labeling

•         Prepare cDNA by reverse transcription by using cyanine 3 (fluoresces green) labeled nucleotides for the healthy cell’s mRNA template and cyanine 5 (fluoresces red) labeled nucleotides for cancerous cell mRNA template. then finally degrade the mRNA.

Hybridization

•        Hybridize the labeled cDNA with DNA probes immobilized on microarray plate.
•        Computer data base having which gene contain in each spot.
•     after hybridizing wash to remove unhybridized cDNA.

Scanning

•         scanner consists with laser, a computer, and a camera.
•         The camera records the images produced when the laser scans the plate. The computer helps in view results & the store data. 

Normalization and analysis

• Green spots → cDNA from healthy tissue hybridized to the target DNA
• Red spots → cDNA from diseased/Cancer tissue hybridized to the target DNA (this is the interest result in disease/cancer diagnosis)
• Yellow spots → both healthy & diseased tissue cDNA hybridized equally to the target DNA (still the gene can carry out is function.
• Black spots → neither healthy or diseased tissue cDNA hybridized to the target DNA   

There are 2 types of DNA Chips/Microarrays - 

1. cDNA based microarray
2. Oligonucleotide based microarray

Applications-

1. Gene expression profiling –  In different cells/tissues, Under different environmental or chemical stimuli, In disease state versus healthy.
2. Discovery of drugs
3. Diagnostics – Microbial identification, microbial genotyping, Antibody detection, Cancer detection
4. Toxicological research (Toxicogenomics)
5. Pharmacogenomics - Individualized medicine.

For more information checkout this video -

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